Several approaches to the photoregulation of proteins have been employed, including photoisomerizable ligands and inhibitors, casting the protein inside a photoisomerizable polymer, and chemical modification of the protein itself .
Several azobenzene protein effectors have been reported to act as photoregulators of enzymes and receptors. Photoregulation of cysteine and serine proteases was carried out using simple photoswitchable inhibitors which block the active site in their trans form only . Conversely, the cis isomers of a-ketoester-based inhibitors were more potent inhibitors of a-chymotrypsin than the trans forms
[138,139]. Screening of a library of photoswitchable peptides selected a single peptide in which the cis and trans isomers bind the nuclear import receptor karyo-pherin a differentially . In fact, photoisomerization of the azobenzene spacer, which acted as a phototelastic linker, regulated the ability of binding sequences to interact with the two distinct karyopherin a subsites simultaneously. A diarylazo derivative bearing an N-ethylmaleimide group on the one extremity and a quaternary ammonium motif on the other was employed for the specific modification of a cysteine residue properly positioned at the edge of the pore of the voltage-gated "Shaker" K+ channel. Photoirradiation of the azo moiety induced the displacement of the ammonium group from the center (E isomer) to the edge (Z isomer) of the pore and thus relieved pore blockade and allowed ion conduction. Reverse CTI upon long-wavelength light irradiation restored the blocked state .
Although the trans to cis photoisomerization of merocyanins leads to a reversible cyclization reaction (see Chapter 2), use of these photoswitchable motifs may be mentioned herein. A photoisomerizable FAD analog was employed to modify glucose oxidase in such a way that electron transfer resulting from glucose oxidation occurs in the spiropyran form and not in the merocyanin form, probably due to the variation of positioning of the FAD moiety .
Covalent incorporation of a photoactivatable molecular switch in proteins can be carried out either via the selective modification of the sequence or by specific derivatization of the active site. Phototunable type S ribonucleases that display enzymatic activity were obtained by noncovalent assembly of the S-protein (resulting from the action of subtilin on ribonuclease A) together with azobenzene derivatives of the complementary S-peptide synthesized chemically bearing a phenyl-azophenylalanine residue at various positions [143,144]. Several azobenzene photoisomerizable amino acids have been synthesized (see Ref. ) such as L-phenylazophenylalanine (Pap or azoAla) 17 which has been inserted inside numerous peptides and proteins, in particular streptavidin, using in vitro protein-synthesizing systems . Pap and a Pap analog were incorporated into a specific position of the dimer interface of the restriction enzyme BamHI, where they blocked the activity in the trans form. Photoirradiation at 366 nm and subsequent CTI induced the specific cleavage of DNA by the enzyme (Fig. 13.20) .
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